Outer Membrane Vesicles (OMVs) are spherical buddings of the outer membrane (OM) that are spontaneously produced by Gram-negative bacteria. They are composed of OM proteins, LPS, phospholipids, and entrapped periplasmic components. Because of their excellent immunostimulatory properties (1-3) and ease of production, OMVs are receiving more and more attention as vaccine candidates. Immunization studies in mice have demonstrated that OMVs can protect against challenges with various pathogenic bacteria (4-12). For Neisseria meningitidis, OMV vaccines have been extensively investigated in clinical trials, and two OMV-based vaccines against Neisseria (MenBvac and MeNZB) are already available for human use (13, 14).
Because of their intrinsic adjuvant properties, the use of OMVs as a delivery vehicle for heterologous antigens has gained considerable interest (15). Several studies have demonstrated that the expression of heterologous antigens in the periplasm or OM of Gram-negative bacteria, by fusion of the heterologous protein to signal peptides or carrier proteins of the host, can lead to their inclusion in OMVs (1-3, 16-19). Importantly, such recombinant OMVs can induce an immune response to the heterologous antigen in immunized mice (2, 3, 12, 17, 18), and even protect them against an otherwise lethal challenge with the pathogen from which the antigen originates (3, 17).
To what extent the specific location of a heterologous antigen within the OMV (periplasm/inside of OM/outside of OM) affects the immune response remains an open question. Theoretically, the outer surface of the OMV appears to be the best option, as this provides the best accessibility for the binding of B-cell receptors (17). There is indeed accumulating evidence that surface exposed antigens evoke superior immune responses (20-24), which makes the precise targeting of heterologous antigens to the OMV surface of special interest.
Various expression systems that specifically target the expression of heterologous proteins to the outer surface of bacterial cells have been developed (see (25-29) for reviews). However, many of these systems can only display small parts of proteins and suffer from low expression levels (30). The two most versatile approaches fuse (parts of) heterologous proteins to Ice Nucleation Protein (25, 31) or autotransporters (21, 32-35) to reach the cell surface. Recently, both systems have also been used to decorate the surface of OMVs with multiple enzymes/antigens (21, 31).
Lipoproteins are membrane-bound proteins that are emerging as key targets for protective immunity, because of their excellent immunostimulatory properties and role as virulence factors. For example, OspA (Borrelia burgdorferi) and fHbp (N. meningitidis) have both been extensively studied as vaccine components against Lyme disease (36-39) and meningitis (40, 41), respectively. Surface expression of heterologous lipoproteins in OMVs has however not been explored so far.
Lipoproteins carry a lipid-modification on their N-terminal cysteine, facilitating the anchoring of hydrophilic proteins in hydrophobic membranes. This highly conserved protein lipidation motif is recognized by the mammalian innate immune system through the Toll like receptor TLR2, providing lipoproteins with superior immunostimulatory properties (44, 45). In Gram-negative bacteria, most lipoproteins are found on the periplasmic side of the inner or outer membrane. They are transferred from the inner membrane to the outer membrane by the Lol (localization of lipoproteins) machinery (46). Lipoproteins that are located on the extracellular side of the outer membrane are less common, and systems or signals guiding transfer over the outer membrane have not yet been elucidated.
In Borrelia, lipoproteins seem to be transferred to the outside of the outer membrane by default, so that the surface of this spirochete is unusually rich in lipoproteins (47). One example of a Borrelia lipoprotein with a surface localization is OspA, for which detailed knowledge regarding its immunogenicity and structure is available because OspA has been extensively investigated as a vaccine component against Lyme disease.
Lyme disease is the most common vector-borne disease in Europe and the United States. Lyme disease is a multisystemic inflammatory disorder that is caused by infection with spirochetes of the B. burgdorferi sensu lato complex as a result of a bite by infected ticks. If an infection is not treated with antibiotics, it can eventually develop into a chronic disease with severe pathology. The only vaccine shortly available for human use (Lymerix) was based on recombinant lipidated OspA. Due to poor sales resulting from claims about auto-immune side-effects, this vaccine was voluntarily withdrawn from the market in 2002, only three years after its introduction (49). However, the side-effect claims were later found to be unsubstantiated (50) and recent Lyme vaccine developments still target OspA, with the much-disputed epitope removed (38, 39).
There is however, still a need in the art for improved vaccine compositions based on Gram-negative outer membrane vesicles displaying antigens of pathogens at their surface, such as Borrelia antigens, and use of these compositions in vaccination e.g. against Lyme disease.